Polymer Derivatives Comprising an Imide Branching Point

ABSTRACT

The invention provides a water-soluble polymer comprising (i) an imide group comprising a linking nitrogen atom, a first carbonyl group covalently attached to the linking nitrogen atom, and a second carbonyl group covalently attached to the linking nitrogen atom; (ii) a first water-soluble polymer segment covalently attached, either directly or through one or more atoms, to the first carbonyl group of the imide group and (iii) a second water-soluble polymer segment covalently attached, either directly or through one or more atoms, to the second carbonyl group of the imide group. The invention also provides, among other things, methods for preparing polymers, conjugates, pharmaceutical compositions and the like.

FIELD OF THE INVENTION

The present invention relates generally to branched polymer derivativeswherein branching in the polymer derivative is effected through an imidemoiety. In addition, the invention relates to conjugates of the polymerderivatives, methods for synthesizing the polymer derivatives andmethods for conjugating the polymer derivatives to active agents andother substances.

BACKGROUND OF THE INVENTION

Scientists and clinicians face a number of challenges in their attemptsto develop active agents that are in a form suitable for delivery to apatient. Active agents that are polypeptides, for example, are oftendelivered via injection rather than orally. In this way, the polypeptideis introduced into the systemic circulation without exposure to theproteolytic environment of the stomach. Injection of polypeptides,however, has several drawbacks. For example, many polypeptides have arelatively short half-life, thereby necessitating repeated injections,which are often inconvenient and painful. Moreover, some polypeptidesmay elicit one or more immune responses with the consequence that thepatient's immune system may be activated to degrade the polypeptide.Thus, delivery of active agents such as polypeptides is oftenproblematic even when these agents are administered by injection.

Some success has been achieved in addressing the problems of deliveringactive agents via injection. For example, conjugating the active agentto a water-soluble polymer has resulted in polymer-active agentconjugates having reduced immunogenicity and antigenicity. In addition,these polymer-active agent conjugates often have greatly increasedhalf-lives compared to their unconjugated counterparts as a result ofdecreased clearance through the kidney and/or decreased enzymaticdegradation in the systemic circulation. As a result of having a greaterhalf-life, the polymer-active agent conjugate requires less frequentdosing, which in turn reduces the overall number of painful injectionsand inconvenient visits with a health care professional. Moreover,active agents that were only marginally soluble demonstrate asignificant increase in water solubility when conjugated to awater-soluble polymer.

Due to its documented safety and approval by the FDA for both topicaland internal use, poly(ethylene glycol) has been conjugated to activeagents. When an active agent is conjugated to polyethylene glycol or“PEG”, the conjugated active agent is conventionally referred to as“PEGylated.” The commercial success of PEGylated active agents, such asPEGASYS® PEGylated interferon alpha-2a (Hoffmann-La Roche, Nutley,N.J.), PEG-INTRON® PEGylated interferon alpha-2b (Schering Corp.,Kennilworth, N.J.), NEULASTA™ PEG-filgrastim (Amgen Inc., Thousand Oaks,Calif.), demonstrates that administration of a conjugated form of anactive agent can have significant advantages over the unconjugatedcounterpart. PEGylated versions of certain small molecules, such asdistearoylphosphatidylethanolamine (Zalipsky (1993) Bioconjug. Chem.4(4):296-299) and fluorouracil (Ouchi et al. (1992) Drug Des. Discov.2(1):93-105), have also been prepared. Harris et al. have provided areview of the effects of PEGylation on pharmaceuticals. Harris et al.(2003) Nat. Rev. Drug Discov. 2(3):214-221.

Despite these successes, conjugation of a polymer to an active agent isoften challenging. For example, attaching a relatively longpoly(ethylene glycol) molecule to an active agent typically impartsgreater water solubility than attaching a shorter poly(ethylene glycol)molecule. However, some conjugates bearing such long poly(ethyleneglycol) moieties have been known to be substantially inactive in vivo.It has been hypothesized that these conjugates are inactive due to thelength of the poly(ethylene glycol) chain, which effectively “wraps”itself around the entire active agent, thereby blocking access topotential ligands required for activity.

The problem associated with inactive conjugates bearing relatively largepoly(ethylene glycol) moieties has been solved, in part, by using“branched” forms of a polymer derivative. “mPEG2-N-hydroxysuccinimide”and “mPEG2-aldehyde,” as shown below, represent examples of branchedversions of a poly(ethylene glycol) derivative.

wherein n represents the number of repeating ethylene oxide monomerunits.

Although solving some of the issues associated with using relativelylarge polymers, branched versions of polymer derivatives also haveproblems. For example, the increased structurally complexity ofbranching often results in a concomitant increase in syntheticcomplexity and/or purification difficulties. As a result, there is anongoing need in the art for more readily synthesized and/or purifiedpolymer derivatives that can be conveniently used in conjugationreactions with active agents.

SUMMARY OF THE INVENTION

In one or more aspects of the invention, a polymer is providedcomprising a first water-soluble polymer segment and a secondwater-soluble polymer segment covalently linked, either directly orthrough a spacer moiety, to an imide moiety. The imide moiety serves asa branching point to which the two polymer segments are attached. Othergroups, such as a reactive group, can also be attached to the imidemoiety. As will be shown in further detail below, the imide moietycomprises two carbonyl groups, each of which is linked to a singlenitrogen atom, herein referred to as a “linking nitrogen atom.”Typically, the first and second water-soluble polymer segments areattached to the carbon atoms of the carbonyl groups, one water-solublepolymer segment per carbonyl group. In embodiments also comprising areactive group, the reactive group is attached to the linking nitrogenatom, either directly or through one or more atoms. Thus, the variouselements are attached in such as way as to result in a branchedstructure.

Attachment of the first and second water-soluble polymer segments, aswell as the reactive group, to the imide moiety can be effected directlyor indirectly. Direct attachment typically comprises a linkage—withoutany intervening atoms—between the imide moiety and the first or secondwater-soluble polymer segment or the reactive group. Indirect attachmentcomprises attachment of the polymer segment or reactive group to theimide moiety through a spacer moiety comprising one or more atoms. Insome instances, both a direct and indirect attachment can be presentwithin a single polymer.

Any water-soluble polymer segment and any reactive group can be used inthe polymer and the invention is not limited in this regard. It ispreferred, however, that the water-soluble polymer segments comprisepoly(ethylene glycol). The polymer segments may include an end-cappingmoiety, such as alkoxy (e.g., methoxy or ethoxy), substituted alkoxy,alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy,aryloxy, substituted aryloxy, or hydroxy. In addition, preferredreactive groups include nucleophiles and electrophiles commonly used insynthetic organic chemistry. Particularly preferred reactive groupsinclude hydroxyl (—OH), ester, orthoester, carbonate, acetal, aldehyde,aldehyde hydrate, ketone, vinyl ketone, ketone hydrate, thione, thionehydrate, hemiketal, sulfur-substituted hemiketal, ketal, alkenyl,acrylate, methacrylate, acrylamide, sulfone, amine, hydrazide, thiol,thiol hydrate, carboxylic acid, isocyanate, isothiocyanate, maleimide

succinimide

benzotriazole

vinylsulfone, chloroethylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates,thiosulfonate, tresylate, and silane. Both water-soluble polymersegments and reactive groups suitable for use in connection with thepresent invention are discussed in more detail below.

In one or more embodiments, the polymer of the invention has one of thefollowing structures:

wherein:

POLY₁ is a first water-soluble polymer segment;

POLY₂ is a second water-soluble polymer segment;

each of (a), (b) and (c) is independently either zero or one;

X₁, when present, is a first spacer moiety;

X₂, when present, is a second spacer moiety;

X₃, when present, is a third spacer moiety; and

Z is a reactive group.

When present, each spacer moiety, X₁, X₂, and X₃, is preferably: abivalent cycloalkyl group comprising 3 to about 20 carbon atoms; anamino acid; a C1-C10 alkylene group; —(CH₂)_(o)—Y—(CH₂)_(p)— wherein oand p are each independently zero to about 10 and Y is selected from thegroup consisting of —O—, —S—, —C(O)—, —C(S)—, —C(O)—O—, —C(O)—NH—,—NH—C(O)—NH—, —O—C(O)—NH—, and —N(R₆)— wherein R₆ is H or an organicradical selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl andsubstituted aryl; or a combination thereof, wherein the spacer moietymay optionally further include an ethylene oxide oligomer chaincomprising 1 to 20 ethylene oxide monomer units.

In one or more aspects, the invention provides a method for preparingthe polymers described herein. Briefly, the method comprises: (i)providing a first water-soluble polymer segment carrying a firstreactive group that comprises a carbonyl group (C═O), (ii) reacting thefirst water-soluble polymer segment with anhydrous ammonia to form awater-soluble polymer intermediate carrying a terminal —C(O)—NH₂ group(iii) treating the water-soluble polymer intermediate with a strong baseto remove a proton from the terminal —C(O)—NH₂ group, (iv) reacting thewater-soluble polymer intermediate with a second water-soluble polymersegment carrying a second reactive group comprising a carbonyl group toform an imide-linked (—C(O)—NH—C(O)—) water-soluble polymer, the imidelinking group comprising a linking nitrogen atom, a first carbonyl groupcovalently attached to the linking nitrogen atom, and a second carbonylgroup covalently attached to the linking nitrogen atom, and (v)optionally, attaching a reactive group to the linking nitrogen atom ofthe imide-linked water-soluble polymer.

In one or more aspects, the invention provides a second method forpreparing the polymers described herein. Briefly, the method comprises:(i) providing a first water-soluble polymer segment carrying a firstreactive group, the first reactive group comprising a carbonyl group,(ii) reacting the first water-soluble polymer segment with a moleculecomprising a second reactive group and an amine group, optionallyseparated by a spacer moiety, to form a water-soluble polymerintermediate comprising a —C(O)—NH— linkage between the water-solublepolymer segment and the second reactive group, (iii) treating thewater-soluble polymer intermediate with a strong base to remove a protonfrom the nitrogen atom of the —C(O)—NH— linkage, and (iv) reacting thewater-soluble polymer intermediate with a second water-soluble polymersegment carrying a third reactive group comprising a carbonyl group toform an imide-linked water-soluble polymer, the imide linking groupcomprising a linking nitrogen atom, a first carbonyl group covalentlyattached to the linking nitrogen atom, and a second carbonyl groupcovalently attached to the linking nitrogen atom.

In one or more aspects of the invention, an active agent-polymerconjugate is provided wherein the conjugate comprises a polymer asdescribed herein covalently attached to an active agent. In addition,the invention also provides a method for forming active-agent conjugateswherein the method comprises the step of contacting a polymer asdescribed herein to a pharmacologically active agent under conditionssuitable to form a covalent attachment between the polymer and thepharmacologically active agent.

In one or more aspects of the invention, pharmaceutical compositions areprovided comprising a polymer conjugate as described herein incombination with a pharmaceutically acceptable carrier. Thepharmaceutical compositions encompass all types of formulations and inparticular those that are suited for injection, e.g., powders that canbe reconstituted as well as suspensions and solutions. Furthermore, theinvention provides a method of treating a patient comprising the step ofadministering a polymer conjugate as described herein.

Additional aspects, advantages, and novel features of the invention willbe set forth in the description that follows, and/or will becomeapparent to one having ordinary skill in the art upon the following, ormay be learned by practice of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to the particularpolymers, synthetic techniques, active agents, and the like as such mayvary.

It must be noted that, as used in this specification and the claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to a“polymer” includes a single polymer as well as two or more of the sameor different polymers, reference to a “conjugate” refers to a singleconjugate as well as two or more of the same or different conjugates,reference to an “excipient” includes a single excipient as well as twoor more of the same or different excipients, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“PEG,” “polyethylene glycol” and “poly(ethylene glycol)” as used herein,are meant to encompass any water-soluble poly(ethylene oxide).Typically, PEGs for use in accordance with the invention comprise thefollowing structure “—O(CH₂CH₂O)_(m)—” where (m) is 2 to 4000. As usedherein, PEG also includes “—CH₂CH₂—O(CH₂CH₂O)_(m)—CH₂CH₂—” and“—(CH₂CH₂O)_(m)—,” depending upon whether or not the terminal oxygenshave been displaced. When the PEG further comprises a spacer moiety (tobe described in greater detail below), the atoms comprising the spacermoiety, when covalently attached to a water-soluble polymer segment, donot result in the formation of an oxygen-oxygen bond (i.e., an “—O—O—”or peroxide linkage). Throughout the specification and claims, it shouldbe remembered that the term “PEG” includes structures having variousterminal or “end capping” groups and so forth. The term “PEG” also meansa polymer that contains a majority, that is to say, greater than 50%, of—CH₂CH₂O— monomeric subunits. With respect to specific forms, the PEGcan take any number of a variety of molecular weights, as well asstructures or geometries such as “branched,” “linear,” “forked,”“multifunctional,” and the like, to be described in greater detailbelow.

The terms “end-capped” or “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group. Thus, examples ofend-capping moieties include alkoxy (e.g., methoxy, ethoxy andbenzyloxy), as well as alkenyloxy, alkynyloxy, aryl, heteroaryl, cyclo,heterocyclo, and the like. In addition, saturated, unsaturated,substituted and unsubstituted forms of each of the foregoing areenvisioned. Moreover, the end-capping group can also be a silane. Theend-capping group can also advantageously comprise a detectable label.When the polymer has an end-capping group comprising a detectable label,the amount or location of the polymer and/or the moiety (e.g., activeagent) of interest to which the polymer is coupled to can be determinedby using a suitable detector. Such labels include, without limitation,fluorescers, chemiluminescers, moieties used in enzyme labeling,colorimetric (e.g., dyes), metal ions, radioactive moieties, and thelike. Suitable detectors include photometers, films, spectrometers, andthe like.

The term “water soluble” as in a “water-soluble polymer segment” and“water-soluble polymer” means any segment or polymer that is soluble inwater at room temperature. Typically, a water-soluble polymer or segmentwill transmit at least about 75%, more preferably at least about 95% oflight, transmitted by the same solution after filtering. On a weightbasis, a water-soluble polymer or segment thereof will preferably be atleast about 35% (by weight) soluble in water, more preferably at leastabout 50% (by weight) soluble in water, still more preferably about 70%(by weight) soluble in water, and still more preferably about 85% (byweight) soluble in water. It is most preferred, however, that thewater-soluble polymer or segment is about 95% (by weight) soluble inwater or completely soluble in water.

The term “non-peptidic” as in a “non-peptidic polymer” refers to apolymer substantially free of peptide linkages. However, the polymer mayinclude a minor number of peptide linkages such as, for example, no morethan about 1 peptide linkage per about 10 monomeric units.

Molecular weight in the context of a water-soluble polymer of theinvention, such as PEG, can be expressed as either a number averagemolecular weight or a weight average molecular weight. Both molecularweight determinations, number average and weight average, can bemeasured using gel permeation chromatography or other liquidchromatography techniques. Other methods for measuring molecular weightvalues can also be used, such as the use of end-group analysis or themeasurement of colligative properties (e.g., freezing-point depression,boiling-point elevation, or osmotic pressure) to determine numberaverage molecular weight or the use of light scattering techniques,ultracentrifugation or viscometry to determine weight average molecularweight. The polymers of the invention are typically polydisperse (i.e.,number average molecular weight and weight average molecular weight ofthe polymers are not equal), possessing low polydispersity values ofpreferably less than about 1.2, more preferably less than about 1.15,still more preferably less than about 1.10, yet still more preferablyless than about 1.05, and most preferably less than about 1.03.

As used herein, the term “carboxylic acid” as in a “carboxylic acid”derivative is a moiety having a

functional group [also represented as a “—COOH” or —C(O)OH]. Unless thecontext clearly dictates otherwise, the term carboxylic acid includesnot only the acid form, but corresponding esters and protected forms aswell. Exemplary protecting groups for carboxylic acids can be found inGreene et al., “PROTECTIVE GROUPS IN ORGANIC SYNTHESIS,” 3^(rd) Edition,John Wiley and Sons, Inc., New York, 1999.

The term “reactive” or “activated” when used in conjunction with aparticular functional group, refers to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react(i.e., a “nonreactive” or “inert” group).

The terms “protected” or “protecting group” or “protective group” referto the presence of a moiety (i.e., the protecting group) that preventsor blocks reaction of a particular chemically reactive functional groupin a molecule under certain reaction conditions. The protecting groupwill vary depending upon the type of chemically reactive group beingprotected as well as the reaction conditions to be employed and thepresence of additional reactive or protecting groups in the molecule, ifany. Protecting groups known in the art can be found in Greene et al.,supra.

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof.

The term “spacer” or “spacer moiety” is used herein to refer to an atomor a collection of atoms optionally used to link one moiety to another,such as a water-soluble polymer segment to a branching group such as animide. The spacer moieties of the invention may be hydrolytically stableor may include a physiologically hydrolyzable or enzymaticallydegradable linkage.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includeethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl when three or more carbon atoms are referenced.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, iso-butyl, and tert-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C₃-C₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl (e.g., 0-2substituted phenyl); substituted phenyl; and the like. “Substitutedaryl” is aryl having one or more non-interfering groups as asubstituent. For substitutions on a phenyl ring, the substituents may bein any orientation (i.e., ortho, meta, or para).

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy,benzyl, etc.), preferably C₁-C₇.

As used herein, “alkenyl” refers to a branched or unbranched hydrocarbongroup of 1 to 15 atoms in length, containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, and the like.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon group of 2 to 15 atoms in length, containing at least onetriple bond, e.g., ethynyl, n-propynyl, n-butynyl, isopentynyl, octynyl,decynyl, and so forth.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof. Heteroaryl rings mayalso be fused with one or more cyclic hydrocarbon, heterocyclic, aryl,or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom which is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from non-interfering substituents.

“Electrophile” refers to an ion or atom or collection of atoms, that maybe ionic, having an electrophilic center, i.e., a center that iselectron seeking, or capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or collection of atoms, that maybe ionic, having a nucleophilic center, i.e., a center that is seekingan electrophilic center or capable of reaction with an electrophile.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively weak bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater will depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include, butare not limited to, carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, ortho esters, peptides andoligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include but are not limited to thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

The terms “active agent,” “biologically active agent” and“pharmacologically active agent” are used interchangeably herein and aredefined to include any agent, drug, compound, composition of matter ormixture that provides some pharmacologic, often beneficial, effect thatcan be demonstrated in-vivo or in vitro. This includes foods, foodsupplements, nutrients, nutriceuticals, drugs, proteins, vaccines,antibodies, vitamins, and other beneficial agents. As used herein, theseterms further include any physiologically or pharmacologically activesubstance that produces a localized or systemic effect in a patient.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a polymer-active agent conjugate—typicallypresent in a pharmaceutical preparation—that is needed to provide adesired level of active agent and/or conjugate in the bloodstream or ina target tissue. The exact amount will depend upon numerous factors,e.g., the particular active agent, the components and physicalcharacteristics of the pharmaceutical preparation, intended patientpopulation, patient considerations, and the like, and can readily bedetermined by one of ordinary skill in the art, based upon theinformation provided herein and available in the relevant literature.

“Multifunctional” in the context of a polymer of the invention means apolymer having 3 or more functional groups contained therein, where thefunctional groups may be the same or different. Multifunctional polymersof the invention will typically contain from about 3-100 functionalgroups, e.g., from 3-50 functional groups, from 3-25 functional groups,from 3-15 functional groups, and from 3 to 10 functional groups i.e., 3,4, 5, 6, 7, 8, 9 or 10 functional groups) within the polymer. A“difunctional” polymer means a polymer having two functional groupscontained therein, either the same (i.e., homodifunctional) or different(i.e., heterodifunctional).

“Branched,” in reference to the geometry or overall structure of apolymer, refers to a polymer having 2 or more polymer “arms.” A branchedpolymer may possess 2 polymer arms, 3 polymer arms, 4 polymer arms, 6polymer arms, 8 polymer arms or more. One particular type of highlybranched polymer is a dendritic polymer or dendrimer, which, for thepurposes of the invention, is considered to possess a structure distinctfrom that of a branched polymer.

A “dendrimer” or dendritic polymer is a globular, size monodispersepolymer in which all bonds emerge radially from a central focal point orcore with a regular branching pattern and with repeat units that eachcontribute a branch point. Dendrimers exhibit certain dendritic stateproperties such as core encapsulation, making them unique from othertypes of polymers.

A basic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate as provided herein, and includes both humans and animals.

“Optional” and “optionally” mean that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

As used herein, the “halo” designator (e.g., fluoro, chloro, iodo,bromo, and so forth) is generally used when the halogen is attached to amolecule, while the suffix “ide” (e.g., fluoride, chloride, iodide,bromide, and so forth) is used when the ionic form is used when thehalogen exists in its independent ionic form (e.g., such as when aleaving group leaves a molecule).

In the context of the present discussion, it should be recognized thatthe definition of a variable provided with respect to one structure orformula is applicable to the same variable repeated in a differentstructure, unless the context dictates otherwise. Thus, for example, thedefinition of “POLY,” “a spacer moiety,” and so forth with respect to apolymer can be equally applicable to a water-soluble polymer conjugateprovided herein.

Turning to a first aspect of the invention, a polymer is providedcomprising an imide moiety comprising the following structure:

As shown, the imide moiety comprises a central linking nitrogen atomthat is covalently bonded to the carbon atoms of two carbonyl groups.

In addition to the imide moiety, the polymer of the invention alsocomprises a first water-soluble polymer segment and a secondwater-soluble polymer segment. Each water-soluble polymer segment isattached, either directly or through a spacer moiety, to one of the twocarbonyl groups attached to the linking nitrogen atom in the imidemoiety, one water-soluble polymer segment per carbonyl group. Thus, thefirst and second water-soluble polymer segments are attached to theimide moiety as shown below.

wherein:

POLY₁ is a first water-soluble polymer segment;

POLY₂ is a second water-soluble polymer segment;

each of (a) and (b) is independently either zero or one;

X₁, when present, is a first spacer moiety; and

X₂, when present, is a second spacer moiety.

Each of the first and second water-soluble polymer segments can compriseany polymer so long as the polymer is water-soluble. Moreover, awater-soluble polymer segment as used herein is typically non-peptidic.Although preferably a poly(ethylene glycol), (i.e., “PEG” or a “PEGpolymer”) a water-soluble polymer segment for use herein can be, forexample, other poly(alkylene glycols) [e.g., poly(propylene glycol)(“PPG”), copolymers of ethylene glycol and propylene glycol and thelike], poly(olefinic alcohol), poly(vinyl pyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol),polyphosphazene, polyoxazoline, poly(oxyethylated polyol), andpoly(N-acryloylmorpholine) such as described in U.S. Pat. No. 5,629,384.The polymer segments can be homopolymers of any of the foregoingpolymers, random copolymers, block copolymers, alternating copolymers,random tripolymers, block tripolymers, alternating tripolymers and thelike. In addition, a water-soluble polymer segment is often linear, butcan be in other forms (e.g., branched, forked, and the like) as will bedescribed in further detail below. In the context of being presentwithin an overall structure, a water-soluble polymer segment has from 1to about 300 termini.

Each water-soluble polymer segment in the overall structure can be thesame or different. It is preferred, however, that all water-solublepolymer segments in the overall structure are of the same type. Forexample, it is preferred that all of the water-soluble polymer segmentswithin a given structure are poly(ethylene glycol).

Although the number average molecular weight and weight averagemolecular weight of any individual water-soluble polymer segment canvary, both molecular weight values will typically be in one or more ofthe following ranges: from about 100 Daltons to about 100,000 Daltons;from about 500 Daltons to about 80,000 Daltons; from about 1,000 Daltonsto about 50,000 Daltons; from about 2,000 Daltons to about 25,000Daltons; from about 5,000 Daltons to about 20,000 Daltons. Exemplarynumber average molecular weights and weight average molecular weightsfor the water-soluble polymer segment include about 1,000 Daltons, about5,000 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000Daltons, about 25,000 Daltons, and about 30,000 Daltons.

Each water-soluble polymer segment is typically biocompatible andnon-immunogenic. With respect to biocompatibility, a substance isconsidered biocompatible if the beneficial effects associated with useof the substance alone or with another substance (e.g., an active agent)in connection with living tissues (e.g., administration to a patient)outweighs any deleterious effects as evaluated by a clinician, e.g., aphysician. With respect to non-immunogenicity, a substance is considerednon-immunogenic if use of the substance alone or with another substancein connection with living tissues does not produce an immune response(e.g., the formation of antibodies) or, if an immune response isproduced, that such a response is not deemed clinically significant orimportant as evaluated by a clinician. It is particularly preferred thatthe polymers and water-soluble polymer segments, described herein aswell as conjugates of active agents and the polymers are biocompatibleand non-immunogenic.

In one form useful in the present invention, free or nonbound PEG is alinear polymer terminated at each end with hydroxyl groups:

HO—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—OH

wherein (m′) typically ranges from zero to about 4,000, preferably fromabout 20 to about 1,000.

The above polymer, alpha-,omega-dihydroxylpoly(ethylene glycol), can berepresented in brief form as HO-PEG-OH where it is understood that the-PEG- symbol represents the following structural unit:

—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—

where (m′) is as defined above.

Another type of free or nonbound PEG useful in the present invention ismethoxy-PEG-OH, or MPEG in brief, in which one terminus is therelatively inert methoxy group, while the other terminus is a hydroxylgroup. The structure of MPEG is given below.

CH₃O—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—OH

where (m′) is as described above.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, can also be used as the PEG polymer. For example,PEG can have the structure:

wherein:

poly_(a) and poly_(b) are PEG backbones (either the same or different),such as methoxy poly(ethylene glycol);

R″ is a nonreactive moiety, such as H, methyl or a PEG backbone; and

P and Q are nonreactive linkages. In a preferred embodiment, thebranched PEG polymer is methoxy poly(ethylene glycol) disubstitutedlysine.

In addition, the PEG can comprise a forked PEG. An example of a free ornonbound forked PEG is represented by the following structure:

wherein: X is a spacer moiety and each Z is an activated terminal grouplinked to CH by a chain of atoms of defined length. U.S. Pat. No.6,362,254 discloses various forked PEG structures capable of use in thepresent invention. The chain of atoms linking the Z functional groups tothe branching carbon atom serve as a tethering group and may comprise,for example, alkyl chains, ether chains, ester chains, amide chains andcombinations thereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups, such as carboxyl, covalently attached along the length of thePEG rather than at the end of the PEG chain. The pendant reactive groupscan be attached to the PEG directly or through a spacer moiety, such asan alkylene group.

In addition to the above-described forms of PEG, the polymer can also beprepared with one or more weak or degradable linkages in the polymer,including any of the above described polymers. For example, PEG can beprepared with ester linkages in the polymer that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:

-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone, include carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al. (1997) Polymer Preprints 38(1):582-3); phosphateester linkages formed, for example, by reacting an alcohol with aphosphate group; hydrazone linkages which are typically formed byreaction of a hydrazide and an aldehyde; acetal linkages that aretypically formed by reaction between an aldehyde and an alcohol; orthoester linkages that are, for example, formed by reaction between aformate and an alcohol; amide linkages formed by an amine group, e.g.,at an end of a polymer such as PEG, and a carboxyl group of another PEGchain; urethane linkages formed from reaction of, e.g., a PEG with aterminal isocyanate group and a PEG alcohol; and oligonucleotidelinkages formed by, for example, a phosphoramidite group, e.g., at theend of a polymer, and a 5′ hydroxyl group of an oligonucleotide.

It is understood by those of ordinary skill in the art that the termpoly(ethylene glycol) or PEG represents or includes all the above formsof PEG.

Those of ordinary skill in the art will recognize that the foregoingdiscussion concerning substantially water-soluble polymers is by nomeans exhaustive and is merely illustrative, and that all polymericmaterials having the qualities described above are contemplated. As usedherein, the “term water-soluble polymer” generally refers to an entiremolecule, which can comprise functional groups such as hydroxyl groups,thiol groups, ortho ester functionalities and so forth. The termwater-soluble polymer segment is generally reserved for use indiscussing specific molecular structures wherein the polymer is but onepart of the overall molecular structure.

As depicted in the above formula, each water-soluble polymer segment isoptionally attached to the rest of the structure through a spacermoiety. A spacer moiety is any atom or series of atoms connecting onepart of a molecule to another. For purposes of the present disclosure,however, a series of atoms is not a spacer moiety when the series ofatoms is immediately adjacent to a polymer and the series of atoms isbut another monomer or oligomer such that the proposed spacer moietywould represent a mere extension of the polymer chain. For example,given the partial structure “POLY-X—,” and POLY is defined as“CH₃O(CH₂CH₂O)_(m)—” wherein (m) is 2 to 4000 and X is defined as aspacer moiety, the spacer moiety cannot be defined as “—CH₂CH₂O—” sincesuch a definition would merely represent an extension of the polymer. Insuch a case, however, an acceptable spacer moiety could be defined as“—CH₂CH₂—.”

Each spacer moiety described herein, such as X₁ and X₂, is preferably: abivalent cycloalkyl group comprising 3 to about 20 carbon atoms; anamino acid; a C1-C10 alkylene group; —(CH₂)_(o)—Y—(CH₂)_(p)— wherein oand p are each independently zero to about 10 and Y is selected from thegroup consisting of —O—, —S—, —C(O)—, —C(S)—, —C(O)—O—, —C(O)—NH—,—NH—C(O)—NH—, —O—C(O)—NH—, and —N(R₆)— wherein R₆ is H or an organicradical selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl andsubstituted aryl; and any combinations of two or more of theabove-described spacer moieties. Additionally, any of the above spacermoieties may further include an ethylene oxide oligomer chain comprising1 to 20 ethylene oxide monomer units, although as noted above, theoligomer chain would not be considered part of the spacer moiety if theoligomer is adjacent to a polymer segment and merely represents andextension of the polymer segment. Particularly preferred examples ofspacer moieties include, but are not limited to, —C(O)—, —C(O)—NH—,—NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—,—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—, —CH₂—C(O)—O—CH₂—,—CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—(CH₂)_(h)—(OCH₂CH₂)_(j)—,—NH—C(O)—O—(CH₂)_(h)—(OCH₂CH₂)_(j)—, —O—, —S—, —N(R₆)—, and combinationsof two or more of any of the foregoing, wherein (h) is zero to six, and(j) is zero to 20. Other specific spacer moieties have the followingstructures: —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, —NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—,and —O—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, wherein the subscript values followingeach methylene indicate the number of methylenes contained in thestructure, e.g., (CH₂)₁₋₆ means that the structure can contain 1, 2, 3,4, 5 or 6 methylenes. Additionally, any of the above spacer moieties mayfurther include an ethylene oxide oligomer chain comprising 1 to 20ethylene oxide monomer units, although as noted above, the oligomerchain would not be considered part of the spacer moiety if the oligomeris adjacent to a polymer segment and merely represents and extension ofthe polymer segment.

In the present context of an amino acid being included in the structuresprovided herein, it should be remembered that the amino acid isconnected to the rest of the structure via one, two, three or moresites. For example, a spacer moiety can result when an amino acid isattached to the rest of the molecule via two covalent attachments. Inaddition, a branching structure can result when an amino acid isattached to the rest of the molecule via three sites. Thus, the aminoacid structure necessarily changes somewhat due to the presence of oneor more covalent attachments (e.g., removal of a hydrogen atom from theamino acid in order to accommodate a covalent linkage). Consequently,reference to an “amino acid” therefore includes the amino acidcontaining one or more linkages to other atoms. The amino acid can beselected from the group consisting of alanine, arginine, asparagines,aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine. Both the D and L forms ofthe amino acids are contemplated.

Each spacer moiety, when present, in the overall structure can be thesame or different than any other spacer moiety in the overall structure.With respect to X₁ and X₂, it is preferred that X₁ and X₂ are the samewhen both are present in the polymer. As depicted in the formula, thelinking moiety “X₁” is present when (a) is defined as one and absentwhen (a) is defined as zero. Similarly, the linking moiety “X₂” ispresent when (b) is defined as one and absent when (b) is defined aszero. Preferred spacer moieties corresponding to X₁ and/or X₂ include—CH₂—, —CH₂CH₂—, and —CH₂CH₂CH₂—.

In embodiments of the polymer of the invention where the linkingnitrogen atom is not covalently attached to a reactive group, thepolymer typically has the following structure:

wherein POLY₁, POLY₂, (a), (b), X₁ (when present), and X₂ (when present)are as previously defined.

In embodiments of the polymer of the invention that further comprise areactive group, the polymer will typically comprise the followingstructure:

wherein:

POLY₁, POLY₂, (a), (b), X₁ (when present), and X₂ (when present) are aspreviously defined;

X³, when present, is a third spacer moiety;

(c) is either zero or one; and

Z is a reactive group.

The third spacer moiety, X₃, when present, can be any atom or series ofatoms connecting one part of a molecule to another. Although the thirdspacer moiety is typically different from the first and/or second spacermoieties (when present), the third spacer moiety can be the same asanother spacer moiety present in the polymer. The third spacer moietycan be selected from one of the group of spacer moieties provided abovewith respect to first and second spacer moieties. The third spacermoiety is absent when (c) is defined as zero and is present when (c) isdefined as one.

A preferred polymer of the invention wherein each of POLY₁ and POLY₂ isdefined as an MPEG has the structure:

wherein each (a), (b), (c), X₁ (when present), X₂ (when present), X₃(when present), and Z are as previously defined, and each m is from 2 toabout 4000. PEG versions other than those that are end-capped withmethoxy (e.g., end-capped with a hydroxyl or other alkoxy such asbenzyloxy) are also envisioned.

In one preferred embodiment of the structure of Formula (III), each of(a) and (b) are defined as zero, resulting in a polymer comprising thefollowing structure:

wherein each of (m), (c), X₃ (when present), and Z are as previouslydefined. Again, PEGs having end-capping groups other than methoxy can beused.

As noted, the polymer may comprise a reactive moiety designated as “Z”throughout the formulae. Preferred reactive moieties are selected fromthe group of electrophiles and nucleophiles. Exemplary reactive groupsinclude hydroxyl (—OH), ester, orthoester, carbonate, acetal, aldehyde,aldehyde hydrate, ketone, vinyl ketone, ketone hydrate, thione, thionehydrate, hemiketal, sulfur-substituted hemiketal, ketal, alkenyl,acrylate, methacrylate, acrylamide, sulfone, amine, hydrazide, thiol,disulfide, thiol hydrate, carboxylic acid, isocyanate, isothiocyanate,maleimide

succinimide

benzotriazole

vinylsulfone, chloroethylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates,thiosulfonate, tresylate, and silane. Specific examples of preferredreactive groups include amine, carboxylic acid, ester, aldehyde, acetal,succinimide, and maleimide.

Illustrative examples of X₃ and Z combinations include

wherein r is 1-5, r′ is 0-5, and R₇ is aryl or alkyl.

Thus, the polymers of the invention comprise many forms. Exemplaryversions of the polymers of the present invention include the following:

wherein each (m) is as previously defined and R is an organic group,preferably C1-C6 alkyl. Again, PEGs having end-capping groups other thanmethoxy can be used.

The polymers of the invention can be prepared by one of ordinary skillin the art based upon reading the present disclosure. Further, thepolymers of the invention can be prepared in any number of ways.Consequently, the polymers provided herein are not limited to thespecific technique or approach used in their preparation. Preferredapproaches, however, will be discussed in detail below.

In one approach for providing the polymers of the invention, the twopolymer segments are linked to the imide group prior to attachment of areactive group to the linking nitrogen atom. In this embodiment, themethod comprises the steps of (i) providing a first water-solublepolymer segment carrying a first reactive group that comprises acarbonyl group (C═O), (ii) reacting the first water-soluble polymersegment with anhydrous ammonia to form a water-soluble polymerintermediate carrying a terminal —C(O)—NH₂ group, (iii) treating thewater-soluble polymer intermediate with a strong base to remove a protonfrom the terminal —C(O)—NH₂ group, (iv) reacting the water-solublepolymer intermediate with a second water-soluble polymer segmentcarrying a second reactive group comprising a carbonyl group to form awater-soluble polymer of the invention. As previously stated, thewater-soluble polymer of the invention comprises (i) an imide groupcomprising a linking nitrogen atom, a first carbonyl group covalentlyattached to the linking nitrogen atom, and a second carbonyl groupcovalently attached to the linking nitrogen atom; (ii) a firstwater-soluble polymer segment covalently attached, either directly orthrough one or more atoms, to the first carbonyl group of the imidegroup; and (iii) a second water-soluble polymer segment covalentlyattached, either directly or through one or more atoms, to the secondcarbonyl group of the imide group. Optionally, the method furthercomprises the step of attaching a reactive group to the linking nitrogenatom of the imide-linked water-soluble polymer.

In the method described immediately above, a protected or derivatizedform of ammonia optionally can be used in place of anhydrous ammonia.When such a protected or derivatized form of ammonia is used, however,the method further includes performing a deprotecting or de-derivatizingstep prior to carrying out any further synthetic steps.

In one embodiment, the first and second carbonyl-containing reactivegroups carried by the water-soluble polymer segments, which may be thesame or different, are each carbonate-containing reactive groups havingthe structure —O—C(O)—O—X_(r), wherein X_(r) is aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocycle, or substitutedheterocycle. Exemplary X_(r) moieties include 1-benzotriazolyl, o-, m-,or p-nitrophenyl, and N-succinimidyl.

Preferred water-soluble polymer segments carrying a reactive group thatcomprises a carbonyl group (C═O) include MPEG terminating inbenzotriazole carbonate (“mPEG-BTC”), MPEG terminating in thesuccinimidyl ester of propionic acid (“mPEG-SPA”), mPEG terminating inthe succinimidyl ester of butanoic acid (“mPEG-SBA”), mPEG terminatingin a thioester (“mPEG-OPTE”), and mPEG terminating in a double ester(“mPEG-CM-HBA-NHS”). Each of these polymers is available from NektarTherapeutics (Huntsville, Ala.).

As noted, the method may include the step of attaching a reactive group,which is preferably accomplished by treating the imide-linkedwater-soluble polymer [from step (iv), above] with a strong base toremove a proton from the linking nitrogen atom and reacting theimide-linked water-soluble polymer with a molecule having the structureZ-(X₃)_(c)-Q, wherein Z is a reactive group, (c) is either zero or one,X₃, when present, is a spacer moiety, and Q is a leaving group. Notethat X, (c), and X₃ and correspond to the moieties carrying the samedesignation in Formulas (II)-(IV) above. Exemplary leaving groupsinclude halo and sulfonate esters. Among halo moieties, bromo, chloro,and iodo are preferred, with bromo and chloro being particularlypreferred. With respect to sulfonate esters, methanesulfonate(abbreviated “Ms”), trifluoromethanesulfonate,trichloromethanesulfonate, 2,2,2-trifluoroethanesulfonate,2,2,2-trichloroethanesulfonate, nonafluorobutanesulfonate,p-bromobenzenesulfonate, p-nitrobenzenesulfonate, and p-toluenesulfonateare particularly preferred, although other sulfonate esters andsimilarly constituted leaving groups known to those of ordinary skill inthe art can be used as well. The presence of the leaving group allowsthe linking nitrogen atom to react with the molecule in a nucleophilicsubstitution reaction, resulting in covalent attachment of the Zreactive group (along with the X₃ spacer when present) to theimide-linked polymer.

An exemplary reaction scheme utilizing the above-described method isshown below as Reaction Scheme I. As shown, an MPEG molecule carrying abenzotriazolyl carbonate reactive group is reacted with anhydrousammonia to form a polymer intermediate carrying a terminal —C(O)—NH₂group. A proton is removed from the terminal —C(O)—NH₂ group using astrong base and a second polymer segment carrying a benzotriazolylcarbonate reactive group is reacted with the polymer intermediate toform an imide-linked polymer. Following a second treatment with a strongbase, a reactive group is attached to the linking nitrogen atom. In thiscase, the terminal reactive group is an ortho ester that can be readilyconverted by hydrolysis to the corresponding carboxylic acid. Thespecific reactive groups and polymer segments used in this reactionscheme could be changed without departing from the invention asexplained above.

In another approach for providing polymers of the invention, a reactivegroup is attached to a first polymer segment prior to attaching thesecond polymer segment to form the imide-linked polymer.

In this embodiment, the method comprises: (i) providing a firstwater-soluble polymer segment carrying a first reactive group, the firstreactive group comprising a carbonyl group, (ii) reacting the firstwater-soluble polymer segment with a molecule comprising a secondreactive group and an amine group, optionally separated by a spacermoiety, to form a water-soluble polymer intermediate comprising a—C(O)—NH— linkage between the water-soluble polymer segment and thesecond reactive group, (iii) treating the water-soluble polymerintermediate with a strong base to remove a proton from the nitrogenatom of the —C(O)—NH— linkage, and (iv) reacting the water-solublepolymer intermediate with a second water-soluble polymer segmentcarrying a third reactive group comprising a carbonyl group to form animide-linked water-soluble polymer, the imide linking group comprising alinking nitrogen atom, a first carbonyl group covalently attached to thelinking nitrogen atom, and a second carbonyl group covalently attachedto the linking nitrogen atom.

Preferably, the molecule comprising a second reactive group and an aminegroup has the structure Z-(X₃)_(c)—NH₂, wherein Z is the second reactivegroup, (c) is either zero or one, and X₃, when present, is a spacermoiety. Note that X, (c), and X₃ and correspond to the moieties carryingthe same designation in Formulas (II)-(IV) above.

An exemplary reaction scheme utilizing the above-described method isshown below as Reaction Scheme II. As shown, an MPEG molecule having aterminal benzotriazolyl carbonate group is reacted with a moleculecarrying a terminal amine group and a second reactive group, which inthis case is an acetal group where R is an organic radical such asalkyl. The resulting polymer is treated with a strong base and reactedwith a second benzotriazolyl carbonate terminated mPEG to form animide-linked polymer carrying an acetal group. The acetal group can thenbe derivatized as desired, such as by hydrolysis to form thecorresponding aldehyde.

The strong base used in the methods described above can be any strongbase known in the art. Preferably, the base is an alkali metal hydroxideor alkali metal hydride (e.g., NaOH or NaH).

For any given polymer, the methods described above advantageouslyprovide the ability to further transform the polymer (either prior orsubsequent to any deprotection step) so that it bears a specificreactive group. Thus, using techniques well known in the art, thepolymer can be functionalized to include a reactive group (e.g., activeester, thiol, maleimide, aldehyde, ketone, and so forth).

For example, when the polymer bears a carboxylic acid as the reactivegroup, the corresponding ester can be formed using conventionaltechniques. For example, the carboxylic acid can undergo acid-catalyzedcondensation with an alcohol, thereby providing the corresponding ester.One approach to accomplish this is to use the method commonly referredto as a Fischer esterification reaction. Other techniques for forming adesired ester are known by those of ordinary skill in the art.

In addition, polymers bearing a carboxylic acid can be modified to formuseful reactive groups other than esters. For example, the carboxylicacid can be further derivatized to form acyl halides, acylpseudohalides, such as acyl cyanide, acyl isocyanate, and acyl azide,neutral salts, such as alkali metal or alkaline-earth metal salts (e.g.calcium, sodium, and barium salts), esters, anhydrides, amides, imides,hydrazides, and the like. In a preferred embodiment, the carboxylic acidis esterified to form an N-succinimidyl ester, o-, m-, or p-nitrophenylester, 1-benzotriazolyl ester, imidazolyl ester, or N-sulfosuccinimidylester. For example, the carboxylic acid can be converted into thecorresponding N-succinimidyl ester by reacting the carboxylic acid withdicyclohexyl carbodiimide (DCC) or diisopropyl carbodiimide (DIC) in thepresence of a N-hydroxysuccinimide.

The steps of the synthesis methods described above take place in anappropriate solvent. One of ordinary skill in the art can determinewhether any specific solvent is appropriate for any given reaction.Typically, however, the solvent is a nonpolar solvent or a polar aproticsolvent. Nonlimiting examples of nonpolar solvents include benzene,xylene, dioxane, tetrahydrofuran (THF), and toluene. Exemplary polaraprotic solvents include, but are not limited to, acetonitrile, DMSO(dimethyl sulfoxide), HMPA (hexamethylphosphoramide), DMF(dimethylformamide), DMA (dimethylacetamide), and NMP(N-methylpyrrolidinone).

The method of preparing the polymers optionally comprises an additionalstep of isolating and recovering the polymer once it is formed. Knownmethods can be used to isolate the polymer, but it is particularlypreferred to use chromatography, e.g., size exclusion chromatography.Alternately or in addition, the method includes the step of purifyingthe polymer once it is formed. Again, standard art-known purificationmethods can be used to purify the polymer.

The polymers of the invention can be stored under an inert atmosphere,such as under argon or under nitrogen. In this way, potentiallydegradative processes associated with, for example, atmospheric oxygen,are reduced or avoided entirely. In some cases, to avoid oxidativedegradation, antioxidants, such as butylated hydroxyl toluene (BHT), canbe added to the final product prior to storage. In addition, it ispreferred to minimize the amount of moisture associated with the storageconditions to reduce potentially damaging reactions associated withwater. Moreover, it is preferred to keep the storage conditions dark inorder to prevent certain degradative processes that involve light. Thus,preferred storage conditions include one or more of the following:storage under dry argon or another dry inert gas; storage attemperatures below about −15° C.; storage in the absence of light; andstorage with a suitable amount (e.g., about 50 to about 500 parts permillion) of an antioxidant such as BHT.

The above-described polymers are useful for conjugation to biologicallyactive agents or surfaces comprising at least one group suitable forreaction with the reactive group on the polymer. For example, aminogroups (e.g., primary amines), hydrazines, hydrazides, and alcohols onan active agent or surface will react with a carboxylic acid group onthe polymer. In addition, a more “activated” version of the carboxylicacid of the polymer can be prepared in order to enhance reactivity tothe biologically active agent or surface. Methods for activatingcarboxylic acids are known in the art and include, for example, themethod for forming an active ester described above. Other approaches foractivating a carboxylic acid are known to those of ordinary skill in theart.

A conjugate of the invention comprises: (i) an imide group comprising alinking nitrogen atom, a first carbonyl group covalently attached to thelinking nitrogen atom, and a second carbonyl group covalently attachedto the linking nitrogen atom; (ii) a first water-soluble polymer segmentcovalently attached, either directly or through one or more atoms, tothe first carbonyl group of the imide group; (iii) a secondwater-soluble polymer segment covalently attached, either directly orthrough one or more atoms, to the second carbonyl group of the imidegroup; and (iv) a pharmacologically active agent covalently attached,either directly or through one or more atoms, to the linking nitrogenatom.

In one embodiment, the conjugate of the invention has the structure:

wherein POLY₁, POLY₂, (a), (b), (c), X₁ (when present), X₂ (whenpresent), and X₃ (when present) are as defined above, and “Active agent”represents a residue of a pharmacologically active agent.

Typically, the polymer is added to the active agent or surface at anequimolar amount (with respect to the desired number of groups suitablefor reaction with the reactive group) or at a molar excess. For example,the polymer can be added to the target active agent at a molar ratio ofabout 1:1 (polymer:active agent), 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 8:1,or 10:1. The conjugation reaction is allowed to proceed untilsubstantially no further conjugation occurs, which can generally bedetermined by monitoring the progress of the reaction over time.Progress of the reaction can be monitored by withdrawing aliquots fromthe reaction mixture at various time points and analyzing the reactionmixture by SDS-PAGE or MALDI-TOF mass spectrometry or any other suitableanalytical method. Once a plateau is reached with respect to the amountof conjugate formed or the amount of unconjugated polymer remaining, thereaction is assumed to be complete. Typically, the conjugation reactiontakes anywhere from minutes to several hours (e.g., from 5 minutes to 24hours or more). The resulting product mixture is preferably, but notnecessarily, purified to separate out excess reagents, unconjugatedreactants (e.g., active agent), undesired multi-conjugated species, andfree or unreacted polymer. The resulting conjugates can then be furthercharacterized using analytical methods such as MALDI, capillaryelectrophoresis, gel electrophoresis, and/or chromatography.

With respect to polymer-active agent conjugates, the conjugates can bepurified to obtain/isolate different conjugated species. Alternatively,and more preferably for lower molecular weight (e.g., less than about 20kiloDaltons, more preferably less than about 10 kiloDaltons) polymers,the product mixture can be purified to obtain the distribution ofwater-soluble polymer segments per active agent. For example, theproduct mixture can be purified to obtain an average of anywhere fromone to five PEGs per active agent (e.g., protein), typically an averageof about three PEGs per active agent (e.g., protein). The strategy forpurification of the final conjugate reaction mixture will depend upon anumber of factors, including, for example, the molecular weight of thepolymer employed, the particular active agent, the desired dosingregimen, and the residual activity and in vivo properties of theindividual conjugate(s).

If desired, conjugates having different molecular weights can beisolated using gel filtration chromatography. That is to say, gelfiltration chromatography is used to fractionate differently numberedpolymer-to-active agent ratios (e.g., 1-mer, 2-mer, 3-mer, and so forth,wherein “1-mer” indicates 1 polymer to active agent, “2-mer” indicatestwo polymers to active agent, and so on) on the basis of their differingmolecular weights (where the difference corresponds essentially to theaverage molecular weight of the water-soluble polymer segments). Forexample, in an exemplary reaction where a 100 kDa protein is randomlyconjugated to a PEG alkanoic acid having a molecular weight of about 20kDa, the resulting reaction mixture will likely contain unmodifiedprotein (MW 100 kDa), mono-pegylated protein (MW 120 kDa), di-pegylatedprotein (MW 140 kDa), and so forth. While this approach can be used toseparate PEG and other polymer conjugates having different molecularweights, this approach is generally ineffective for separatingpositional isomers having different polymer attachment sites within theprotein. For example, gel filtration chromatography can be used toseparate mixtures of PEG 1-mers, 2-mers, 3-mers, and so forth, althougheach of the recovered PEG-mer compositions may contain PEGs attached todifferent reactive amino groups (e.g., lysine residues) within theactive agent.

Gel filtration columns suitable for carrying out this type of separationinclude Superdex™ and Sephadex™ columns available from AmershamBiosciences (Piscataway, N.J.). Selection of a particular column willdepend upon the desired fractionation range desired. Elution isgenerally carried out using a suitable buffer, such as phosphate,acetate, or the like. The collected fractions may be analyzed by anumber of different methods, for example, (i) optical density (OD) at280 nm for protein content, (ii) bovine serum albumin (BSA) proteinanalysis, (iii) iodine testing for PEG content [Sims et al. (1980) Anal.Biochem, 107:60-63], and (iv) sodium dodecyl sulfphate polyacrylamidegel electrophoresis (SDS PAGE), followed by staining with barium iodide.

Separation of positional isomers is carried out by reverse phasechromatography using a reverse phase-high performance liquidchromatography (RP-HPLC) C18 column (Amersham Biosciences or Vydac) orby ion exchange chromatography using an ion exchange column, e.g., aSepharose™ ion exchange column available from Amersham Biosciences.Either approach can be used to separate polymer-active agent isomershaving the same molecular weight (positional isomers).

Following conjugation, and optionally additional separation steps, theconjugate mixture can be concentrated, sterile filtered, and stored atlow a temperature, typically from about −20° C. to about −80° C.Alternatively, the conjugate may be lyophilized, either with or withoutresidual buffer and stored as a lyophilized powder. In some instances,it is preferable to exchange a buffer used for conjugation, such assodium acetate, for a volatile buffer such as ammonium carbonate orammonium acetate, that can be readily removed during lyophilization, sothat the lyophilized powder is absent residual buffer. Alternatively, abuffer exchange step may be used using a formulation buffer, so that thelyophilized conjugate is in a form suitable for reconstitution into aformulation buffer and ultimately for administration to a mammal.

The polymers of the invention can be attached, either covalently ornoncovalently, to a number of entities including films, chemicalseparation and purification surfaces, solid supports, metal surfacessuch as gold, titanium, tantalum, niobium, aluminum, steel, and theiroxides, silicon oxide, macromolecules (e.g., proteins, polypeptides, andso forth), and small molecules. Additionally, the polymers can also beused in biochemical sensors, bioelectronic switches, and gates. Thepolymers can also be employed as carriers for peptide synthesis, for thepreparation of polymer-coated surfaces and polymer grafts, to preparepolymer-ligand conjugates for affinity partitioning, to preparecross-linked or non-cross-linked hydrogels, and to preparepolymer-cofactor adducts for bioreactors.

A biologically active agent for use in coupling to a polymer aspresented herein may be any one or more of the following. Suitableagents can be selected from, for example, hypnotics and sedatives,psychic energizers, tranquilizers, respiratory drugs, anticonvulsants,muscle relaxants, antiparkinson agents (dopamine antagnonists),analgesics, anti-inflammatories, antianxiety drugs (anxiolytics),appetite suppressants, antimigraine agents, muscle contractants,anti-infectives (antibiotics, antivirals, antifungals, vaccines)antiarthritics, antimalarials, antiemetics, anepileptics,bronchodilators, cytokines, growth factors, anti-cancer agents,antithrombotic agents, antihypertensives, cardiovascular drugs,antiarrhythimics, antioxicants, anti-asthma agents, hormonal agentsincluding contraceptives, sympathomimetics, diuretics, lipid regulatingagents, antiandrogenic agents, antiparasitics, anticoagulants,neoplastics, antineoplastics, hypoglycemics, nutritional agents andsupplements, growth supplements, antienteritis agents, vaccines,antibodies, diagnostic agents, and contrasting agents.

More particularly, the active agent may fall into one of a number ofstructural classes, including but not limited to small molecules(preferably water insoluble small molecules), peptides, polypeptides,proteins, polysaccharides, steroids, nucleotides, oligonucleotides,polynucleotides, fats, electrolytes, and the like. Preferably, an activeagent for coupling to a polymer as described herein possesses a nativeamino group, or alternatively, is modified to contain at least onereactive amino group suitable for conjugating to a polymer describedherein.

Specific examples of active agents suitable for covalent attachmentinclude, but are not limited to, aspariginase, amdoxovir (DAPD), antide,becaplermin, calcitonins, cyanovirin, denileukin diftitox,erythropoietin (EPO), EPO agonists (e.g., peptides from about 10-40amino acids in length and comprising a particular core sequence asdescribed in WO 96/40749), dornase alpha, erythropoiesis stimulatingprotein (NESP), coagulation factors such as Factor V, Factor VII, FactorVIIa, Factor VIII, Factor IX, Factor X, Factor XII, Factor XIII, vonWillebrand factor; ceredase, cerezyme, alpha-glucosidase, collagen,cyclosporin, alpha defensins, beta defensins, exedin-4, granulocytecolony stimulating factor (GCSF), thrombopoietin (TPO), alpha-1proteinase inhibitor, elcatonin, granulocyte macrophage colonystimulating factor (GMCSF), fibrinogen, filgrastim, growth hormoneshuman growth hormone (hGH), growth hormone releasing hormone (GHRH),GRO-beta, GRO-beta antibody, bone morphogenic proteins such as bonemorphogenic protein-2, bone morphogenic protein-6, OP-1; acidicfibroblast growth factor, basic fibroblast growth factor, CD-40 ligand,heparin, human serum albumin, low molecular weight heparin (LMWH),interferons such as interferon alpha, interferon beta, interferon gamma,interferon omega, interferon tau, consensus interferon; interleukins andinterleukin receptors such as interleukin-1 receptor, interleukin-2,interluekin-2 fusion proteins, interleukin-1 receptor antagonist,interleukin-3, interleukin-4, interleukin-4 receptor, interleukin-6,interleukin-8, interleukin-12, interleukin-13 receptor, interleukin-17receptor; lactoferrin and lactoferrin fragments, luteinizing hormonereleasing hormone (FSH), insulin, pro-insulin, insulin analogues (e.g.,mono-acylated insulin as described in U.S. Pat. No. 5,922,675), amylin,C-peptide, somatostatin, somatostatin analogs including octreotide,vasopressin, follicle stimulating hormone (FSH), influenza vaccine,insulin-like growth factor (IGF), insulintropin, macrophage colonystimulating factor (M-CSF), plasminogen activators such as alteplase,urokinase, reteplase, streptokinase, pamiteplase, lanoteplase, andteneteplase; nerve growth factor (NGF), osteoprotegerin,platelet-derived growth factor, tissue growth factors, transforminggrowth factor-1, vascular endothelial growth factor, leukemia inhibitingfactor, keratinocyte growth factor (KGF), glial growth factor (GGF), TCell receptors, CD molecules/antigens, tumor necrosis factor (TNF),monocyte chemoattractant protein-1, endothelial growth factors,parathyroid hormone (PTH), glucagon-like peptide, somatotropin, thymosinalpha 1, rasburicase, thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta10, thymosin beta 9, thymosin beta 4, alpha-1 antitrypsin,phosphodiesterase (PDE) compounds, VLA-4 (very late antigen-4), VLA-4inhibitors, bisphosphonates, respiratory syncytial virus antibody,cystic fibrosis transmembrane regulator (CFTR) gene, deoxyribonuclease(Dnase), bactericidal/permeability increasing protein (BPI), andanti-CMV antibody. Exemplary monoclonal antibodies include etanercept (adimeric fusion protein consisting of the extracellular ligand-bindingportion of the human 75 kD TNF receptor linked to the Fc portion ofIgG1), abciximab, adalimumab, afelimomab, alemtuzumab, antibody toB-lymphocyte (lymphostat-B™), atlizumab, basiliximab, bevacizumab,biciromab, CAT-213 or bertilimumab, CDP-571, CDP-870, cetuximab,clenoliximab, daclizumab, eculizumab, edrecolomab, efalizumab,epratuzumab, fontolizumab, gavilimomab, gemtuzumab ozogamicin,ibritumomab tiuxetan, infliximab, inolimomab, keliximab, labetuzumab,lerdelimumab, radiolabeled lym-1, metelimumab, mepolizumab, mitumomab,muromonad-CD3, nebacumab, natalizumab, odulimomab, omalizumab,oregovbmab, palivizumab, pemtumomab, pexelizumab, rituximab satumomabpendetide, sevirumab, siplizumab, tositumomab and I¹³¹ tositumomab,olizumab, trastuzumab, tuvirumab, and visilizumab.

Additional agents suitable for covalent attachment include, but are notlimited to, adefovir, alosetron, amifostine, amiodarone, aminocaproicacid, aminohippurate sodium, aminoglutethimide, aminolevulinic acid,aminosalicylic acid, amsacrine, anagrelide, anastrozole, aripiprazole,asparaginase, anthracyclines, bexarotene, bicalutamide, bleomycin,buserelin, busulfan, cabergoline, capecitabine, carboplatin, carmustine,chlorambucin, cilastatin sodium, cisplatin, cladribine, clodronate,cyclophosphamide, cyproterone, cytarabine, camptothecins, 13-cisretinoic acid, all trans retinoic acid; dacarbazine, dactinomycin,daunorubicin, deferoxamine, dexamethasone, diclofenac,diethylstilbestrol, docetaxel, doxorubicin, dutasteride, epirubicin,estramustine, etoposide, exemestane, ezetimibe, fexofenadine,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,fondaparinux, fulvestrant, gamma-hydroxybutyrate, gemcitabine,epinephrine, L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib,irinotecan, itraconazole, goserelin, letrozole, leucovorin, levamisole,lisinopril, lovothyroxine sodium, lomustine, mechlorethamine,medroxyprogesterone, megestrol, melphalan, mercaptopurine, metaraminolbitartrate, methotrexate, metoclopramide, mexiletine, mitomycin,mitotane, mitoxantrone, naloxone, nicotine, nilutamide, nitisinone,octreotide, oxaliplatin, pamidronate, pentostatin, pilcamycin, porfimer,prednisone, procarbazine, prochlorperazine, ondansetron, oxaliplatin,raltitrexed, sirolimus, streptozocin, tacrolimus, pimecrolimus,tamoxifen, tegaserod, temozolomide, teniposide, testosterone,tetrahydrocannabinol, thalidomide, thioguanine, thiotepa, topotecan,treprostinil, tretinoin, valdecoxib, celecoxib, rofecoxib, valrubicin,vinblastine, vincristine, vindesine, vinorelbine, voriconazole,dolasetron, granisetron; formoterol, fluticasone, leuprolide, midazolam,alprazolam, amphotericin B, podophylotoxins, nucleoside antivirals,aroyl hydrazones, sumatriptan; macrolides such as erythromycin,oleandomycin, troleandomycin, roxithromycin, clarithromycin, davercin,azithromycin, flurithromycin, dirithromycin, josamycin, spiromycin,midecamycin, loratadine, desloratadine, leucomycin, miocamycin,rokitamycin, andazithromycin, and swinolide A; fluoroquinolones such asciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin,lomefloxacin, sparfioxacin, temafloxacin, pefloxacin, amifloxacin,fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin,clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin,netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, andstreptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin,daptomycin, gramicidin, colistimethate; polymixins such as polymixin B,capreomycin, bacitracin, penems; penicillins includingpenicllinase-sensitive agents like penicillin G, penicillin V;penicllinase-resistant agents like methicillin, oxacillin, cloxacillin,dicloxacillin, floxacillin, nafcillin; gram negative microorganismactive agents like ampicillin, amoxicillin, and hetacillin, cillin, andgalampicillin; antipseudomonal penicillins like carbenicillin,ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporinslike cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone,cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin,cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil,cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatrizine,cephacetrile, cefepime, cefixime, cefonicid, cefoperazone, cefotetan,cefmetazole, ceftazidime, loracarbef, and moxalactam, monobactams likeaztreonam; and carbapenems such as imipenem, meropenem, and ertapenem,pentamidine isetionate, albuterol sulfate, lidocaine, metaproterenolsulfate, beclomethasone diprepionate, triamcinolone acetamide,budesonide acetonide, fluticasone, ipratropium bromide, flunisolide,cromolyn sodium, and ergotamine tartrate; taxanes such as paclitaxel;SN-38, and tyrphostines.

Preferred small molecules for coupling to a polymer as described hereinare those having at least one naturally occurring amino group. Preferredmolecules such as these include aminohippurate sodium, amphotericin B,doxorubicin, aminocaproic acid, aminolevulinic acid, aminosalicylicacid, metaraminol bitartrate, pamidronate disodium, daunorubicin,levothyroxine sodium, lisinopril, cilastatin sodium, mexiletine,cephalexin, deferoxamine, and amifostine.

Preferred peptides or proteins for coupling to a polymer as describedherein include EPO, IFN-α, IFN-β, consensus IFN, Factor VIII, Factor IX,GCSF, GMCSF, hGH, insulin, FSH, and PTH.

The above exemplary biologically active agents are meant to encompass,where applicable, analogues, agonists, antagonists, inhibitors, isomers,and pharmaceutically acceptable salt forms thereof. In reference topeptides and proteins, the invention is intended to encompass synthetic,recombinant, native, glycosylated, and non-glycosylated forms, as wellas biologically active fragments thereof.

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form. Inone embodiment, the conjugate is formulated with a liquid diluent, suchas bacteriostatic water for injection, dextrose 5% in water,phosphate-buffered saline, Ringer's solution, saline solution, sterilewater, deionized water, or combinations thereof.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases may be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The pharmaceutical preparations encompass all types of formulations andin particular those that are suited for injection, e.g., powders thatcan be reconstituted as well as suspensions and solutions. For example,the pharmaceutical preparation can be in the form of a lyophilized cakeor powder. The amount of the conjugate (i.e., the conjugate formedbetween the active agent and the polymer described herein) in thecomposition will vary depending on a number of factors, but willoptimally be a therapeutically effective dose when the composition isstored in a unit dose container (e.g., a vial). In addition, thepharmaceutical preparation can be housed in a syringe. A therapeuticallyeffective dose can be determined experimentally by repeatedadministration of increasing amounts of the conjugate in order todetermine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about5%-98% by weight, more preferably from about 15-95% by weight of theexcipient, with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andKibbe, A. H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical preparations of the present invention are typically,although not necessarily, administered via injection and are thereforegenerally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

As previously described, the conjugates can be administered injectedparenterally by intravenous injection, or less preferably byintramuscular or by subcutaneous injection. Suitable formulation typesfor parenteral administration include ready-for-injection solutions, drypowders for combination with a solvent prior to use, suspensions readyfor injection, dry insoluble compositions for combination with a vehicleprior to use, and emulsions and liquid concentrates for dilution priorto administration, among others.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with conjugate. The method comprisesadministering, generally via injection, a therapeutically effectiveamount of the conjugate (preferably provided as part of a pharmaceuticalpreparation). The method of administering may be used to treat anycondition that can be remedied or prevented by administration of theparticular conjugate. Those of ordinary skill in the art appreciatewhich conditions a specific conjugate can effectively treat. The actualdose to be administered will vary depend upon the age, weight, andgeneral condition of the subject as well as the severity of thecondition being treated, the judgment of the health care professional,and conjugate being administered. Therapeutically effective amounts areknown to those skilled in the art and/or are described in the pertinentreference texts and literature. Generally, a therapeutically effectiveamount will range from about 0.001 mg to 100 mg, preferably in dosesfrom 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10mg/day to 50 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

One advantage of administering the conjugates of the present inventionis that individual water-soluble polymer portions can be cleaved off.Such a result is advantageous when clearance from the body ispotentially a problem because of the polymer size. Optimally, cleavageof each water-soluble polymer portion is facilitated through the use ofphysiologically cleavable and/or enzymatically degradable linkages suchas carbonate or ester-containing linkages. In this way, clearance of theconjugate (via cleavage of individual water-soluble polymer portions)can be modulated by selecting the polymer molecular size and the typefunctional group that would provide the desired clearance properties.One of ordinary skill in the art can determine the proper molecular sizeof the polymer as well as the cleavable functional group. For example,one of ordinary skill in the art, using routine experimentation, candetermine a proper molecular size and cleavable functional group byfirst preparing a variety of polymer derivatives with different polymerweights and cleavable functional groups, and then obtaining theclearance profile (e.g., through periodic blood or urine sampling) byadministering the polymer derivative to a patient and taking periodicblood and/or urine sampling. Once a series of clearance profiles havebeen obtained for each tested conjugate, a suitable conjugate can beidentified.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the experimental that follow areintended to illustrate and not limit the scope of the invention. Otheraspects, advantages and modifications within the scope of the inventionwill be apparent to those skilled in the art to which the inventionpertains.

All articles, books, patents, patent publications and other publicationsreferenced herein are hereby incorporated by reference in theirentireties.

1. A water-soluble polymer comprising (i) an imide group comprising alinking nitrogen atom, a first carbonyl group covalently attached to thelinking nitrogen atom, and a second carbonyl group covalently attachedto the linking nitrogen atom; (ii) a first water-soluble polymer segmentcovalently attached, either directly or through one or more atoms, tothe first carbonyl group of the imide group; and (iii) a secondwater-soluble polymer segment covalently attached, either directly orthrough one or more atoms, to the second carbonyl group of the imidegroup.
 2. The water-soluble polymer of claim 1, further comprising (iv)a reactive group covalently attached, either directly or through one ormore atoms, to the linking nitrogen atom.
 3. The water-soluble polymerof claims 1 or 2, wherein the first and second water-soluble polymersegments are independently selected from the group consisting ofpoly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol),polyoxazoline, poly(acryloylmorpholine), and poly(oxyethylated polyol).4. The water-soluble polymer of claims 1 or 2, wherein the first andsecond water-soluble polymer segments comprise a poly(alkylene oxide).5. The water-soluble polymer of claim 4, wherein the first and secondwater-soluble polymer segments comprise poly(ethylene glycol).
 6. Thewater-soluble polymer of claim 5, wherein each poly(ethylene glycol) isterminally capped with an end-capping moiety.
 7. The water-solublepolymer of claim 6, wherein each end-capping moiety is independentlyselected from the group consisting alkoxy, substituted alkoxy,alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy,aryloxy, substituted aryloxy, and hydroxy.
 8. The water-soluble polymerof claim 6, wherein each end-capping moiety is alkoxy.
 9. Thewater-soluble polymer of claim 8, wherein each alkoxy is methoxy. 10.The water-soluble polymer of claim 6, wherein the end-capping moiety ishydroxy.
 11. The water-soluble polymer of claim 5, wherein eachpoly(ethylene glycol) has a weight average molecular weight of fromabout 100 Daltons to about 100,000 Daltons.
 12. The water-solublepolymer of claim 11, wherein each poly(ethylene glycol) has a weightaverage molecular weight of from about 500 Daltons to about 80,000Daltons.
 13. The water-soluble polymer of claim 12, wherein eachpoly(ethylene glycol) has a weight average molecular weight of fromabout 1,000 Daltons to about 50,000 Daltons.
 14. The water-solublepolymer of claim 13, wherein each poly(ethylene glycol) has a weightaverage molecular weight of from about 2,000 Daltons to about 25,000Daltons.
 15. The water-soluble polymer of claim 14, wherein eachpoly(ethylene glycol) has a weight average molecular weight of fromabout 5,000 Daltons to about 26,000 Daltons.
 16. The water-solublepolymer of claim 2, wherein the reactive group is selected from thegroup consisting of hydroxyl (—OH), ester, orthoester, carbonate,acetal, aldehyde, aldehyde hydrate, ketone, vinyl ketone, ketonehydrate, thione, thione hydrate, hemiketal, sulfur-substitutedhemiketal, ketal, alkenyl, acrylate, methacrylate, acrylamide, sulfone,amine, hydrazide, thiol, thiol hydrate, carboxylic acid, isocyanate,isothiocyanate, maleimide

succinimide

benzotriazole

vinylsulfone, chloroethylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates,thiosulfonate, tresylate, and silane.
 17. The water-soluble polymer ofclaim 1 or 2, wherein the first water-soluble polymer segment isdirectly attached to the first carbonyl group of the imide group and thesecond water-soluble polymer segment is directly attached to the secondcarbonyl group of the imide group.
 18. The water-soluble polymer ofclaim 1 or 2, wherein at least one of the first and second water-solublepolymer segments is attached to their respective carbonyl group of theimide group through a spacer moiety, wherein the spacer moiety isselected from the group consisting of a bivalent cycloalkyl groupcomprising 3 to about 20 carbon atoms, an amino acid, a C1-C10 alkylenegroup, —(CH₂)_(o)—Y—(CH₂)_(p)— wherein (o) and (p) are eachindependently zero to about 10 and Y is selected from the groupconsisting of —O—, —S—, —N(R₆)— wherein R₆ is H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl and substitutedaryl, —C(O)—, —C(S)—, —C(O)—O—, —C(O)—NH—, —NH—C(O)—NH—, and—O—C(O)—NH—, and combinations thereof, wherein the spacer moietyoptionally further includes an ethylene oxide oligomer chain comprising1 to 20 ethylene oxide monomer units.
 19. The water-soluble polymer ofclaim 2, wherein the reactive group is covalently attached to thelinking nitrogen atom through a spacer moiety, wherein the spacer moietyis selected from the group consisting of a bivalent cycloalkyl groupcomprising 3 to about 20 carbon atoms, an amino acid, a C1-C10 alkylenegroup, —(CH₂)_(o)—Y—(CH₂)_(p)— wherein (o) and (p) are eachindependently zero to about 10 and Y is selected from the groupconsisting of —O—, —S—, —N(R₆)— wherein R₆ is H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl and substitutedaryl, —C(O)—, —C(S)—, —C(O)—O—, —C(O)—NH—, —NH—C(O)—NH—, and—O—C(O)—NH—, and combinations thereof, wherein the spacer moietyoptionally further includes an ethylene oxide oligomer chain comprising1 to 20 ethylene oxide monomer units.
 20. A water-soluble polymer havingone of the following structures:

wherein: POLY₁ is a first water-soluble polymer segment; POLY₂ is asecond water-soluble polymer segment; each of (a), (b) and (c) isindependently either zero or one; X₁, when present, is a first spacermoiety; X₂, when present, is a second spacer moiety; X₃, when present,is a third spacer moiety; and Z is a reactive group.
 21. Thewater-soluble polymer of claim 20, wherein each of POLY₁ and POLY₂ isindependently selected from the group consisting of poly(alkyleneoxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline,poly(acryloylmorpholine), and poly(oxyethylated polyol).
 22. Thewater-soluble polymer of claim 20, wherein POLY₁ and POLY₂ are the same.23. The water-soluble polymer of claim 20, wherein POLY₁ and POLY₂ aredifferent.
 24. The water-soluble polymer of claim 20, wherein each ofPOLY₁ and POLY₂ is a poly(alkylene oxide).
 25. The water-soluble polymerof claim 20, wherein each of POLY₁ and POLY₂ is a poly(ethylene glycol).26. The water-soluble polymer of claim 25, wherein each poly(ethyleneglycol) is terminally capped with an end-capping moiety.
 27. Thewater-soluble polymer of claim 26, wherein each end-capping moiety isindependently selected from the group consisting alkoxy, substitutedalkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substitutedalkynyloxy, aryloxy, substituted aryloxy, and hydroxy.
 28. Thewater-soluble polymer of claim 27, wherein each end-capping moiety isalkoxy.
 29. The water-soluble polymer of claim 28, wherein each alkoxyis methoxy.
 30. The water-soluble polymer of claim 27, wherein eachend-capping moiety is hydroxy.
 31. The water-soluble polymer of claim25, wherein each poly(ethylene glycol) has a weight average molecularweight of from about 100 Daltons to about 100,000 Daltons.
 32. Thewater-soluble polymer of claim 31, wherein each poly(ethylene glycol)has a weight average molecular weight of from about 1,000 Daltons toabout 50,000 Daltons.
 33. The water-soluble polymer of claim 32, whereineach poly(ethylene glycol) has a weight average molecular weight of fromabout 5,000 Daltons to about 20,000 Daltons.
 34. The water-solublepolymer of claim 20, wherein each of POLY₁ and POLY₂ independently has astructure selected from the group consisting of homopolymer, alternatingcopolymer, random copolymer, block copolymer, alternating tripolymer,random tripolymer, and block tripolymer.
 35. The water-soluble polymerof claim 20, wherein each of (a) and (b) is one.
 36. The water-solublepolymer of claim 35, wherein each of X₁ and X₂ is the same.
 37. Thewater-soluble polymer of claim 35, wherein each of X₁ and X₂ isdifferent.
 38. The water-soluble polymer of claim 35, wherein X₁ and X₂are independently selected from the group consisting of a bivalentcycloalkyl group comprising 3 to about 20 carbon atoms, an amino acid, aC1-C10 alkylene group, —(CH₂)_(o)—Y—(CH₂)_(p)— wherein (o) and (p) areeach independently zero to about 10 and Y is selected from the groupconsisting of —O—, —S—, —N(R₆)— wherein R₆ is H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl and substitutedaryl, —C(O)—, —C(S)—, —C(O)—O—, —C(O)—NH—, —NH—C(O)—NH—, and—O—C(O)—NH—, and combinations thereof, wherein X₁ or X₂ may optionallyfurther include an ethylene oxide oligomer chain comprising 1 to 20ethylene oxide monomer units.
 39. The water-soluble polymer of claim 35,wherein each of X₁ and X₂ is independently selected from the groupconsisting of —C(O)—, —C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—,—CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—,—O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—,—CH₂—C(O)—O—CH₂—, —CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—(CH₂)_(h)—(OCH₂CH₂)_(j)—,—NH—C(O)—O—(CH₂)_(h)—(OCH₂CH₂)_(j)—, —C(O)—NH—(CH₂)_(h)—NH—C(O)—,—NH—C(O)—NH—(CH₂)_(h)—NH—C(O)—, —O—C(O)—NH—(CH₂)_(h)—NH—C(O)—, —O—, —S—,—N(R₆)—, and combinations of two or more of any of the foregoing,wherein (h) is zero to six, (j) is zero to 20, and R₆ is H or an organicradical selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl andsubstituted aryl.
 40. The water-soluble polymer of claim 20, wherein (c)is one.
 41. The water-soluble polymer of claim 40, wherein X₃ isselected from the group consisting of a bivalent cycloalkyl groupcomprising 3 to about 20 carbon atoms, an amino acid, a C1-C10 alkylenegroup, —(CH₂)_(o)—Y—(CH₂)_(p)— wherein (o) and (p) are eachindependently zero to about 10 and Y is selected from the groupconsisting of —O—, —S—, —N(R₆)— wherein R₆ is H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl and substitutedaryl, —C(O)—, —C(S)—, —C(O)—O—, —C(O)—NH—, —NH—C(O)—NH—, and—O—C(O)—NH—, and combinations thereof, wherein X₃ may optionally furtherinclude an ethylene oxide oligomer chain comprising 1 to 20 ethyleneoxide monomer units.
 42. The water-soluble polymer of claim 40, whereinX₃ is selected from the group consisting of —C(O)—, —C(O)—NH—,—NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—,—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—, —CH₂—C(O)—O—CH₂—,—CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—(CH₂)_(h)—(CH₂CH₂)_(j)—, —NH—C(O)—O—(CH₂)_(h)—(OCH₂CH₂)_(j)—,—C(O)—NH—(CH₂)_(h)—NH—C(O)—, —NH—C(O)—NH—(CH₂)_(h)—NH—C(O)—,—O—C(O)—NH—(CH₂)_(h)—NH—C(O)—, —O—, —S—, —N(R₆)—, and combinations oftwo or more of any of the foregoing, wherein (h) is zero to six, (j) iszero to 20, and R₆ is H or an organic radical selected from the groupconsisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl and substituted aryl.
 43. Thewater-soluble polymer of claim 20, wherein Z comprises an electrophile.44. The water-soluble polymer of claim 20, wherein Z comprises anucleophile.
 45. The water-soluble polymer of claim 20, wherein Z isselected from the group consisting of hydroxyl (—OH), ester, orthoester,carbonate, acetal, aldehyde, aldehyde hydrate, ketone, vinyl ketone,ketone hydrate, thione, thione hydrate, hemiketal, sulfur-substitutedhemiketal, ketal, alkenyl, acrylate, methacrylate, acrylamide, sulfone,amine, hydrazide, thiol, thiol hydrate, carboxylic acid, isocyanate,isothiocyanate, maleimide

succinimide

benzotriazole

vinylsulfone, chloroethylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates,thiosulfonate, tresylate, and silane.
 46. The polymer of claim 20,selected from the group consisting of

wherein each (m) is independently 2 to 4,000 and R is an organic group,preferably C1-C6 alkyl.
 47. A conjugate comprising: (i) an imide groupcomprising a linking nitrogen atom, a first carbonyl group covalentlyattached to the linking nitrogen atom, and a second carbonyl groupcovalently attached to the linking nitrogen atom; (ii) a firstwater-soluble polymer segment covalently attached, either directly orthrough one or more atoms, to the first carbonyl group of the imidegroup; (iii) a second water-soluble polymer segment covalently attached,either directly or through one or more atoms, to the second carbonylgroup of the imide group; and (iv) a pharmacologically active agentcovalently attached, either directly or through one or more atoms, tothe linking nitrogen atom.
 48. The conjugate of claim 47, having thestructure:

wherein: POLY₁ is a first water-soluble polymer segment; POLY₂ is asecond water-soluble polymer segment; each of (a), (b) and (c) isindependently either zero or one; X₁, when present, is a first spacermoiety; X₂, when present, is a second spacer moiety; X₃, when present,is a third spacer moiety; and Active agent represents a residue of apharmacologically active agent.
 49. A method of preparing a conjugatecomprising the step of contacting a water-soluble polymer according toany one of claims 1-46 with a pharmacologically active agent underconditions suitable to form a covalent attachment between thewater-soluble polymer and the pharmacologically active agent.
 50. Apharmaceutical preparation comprising a conjugate according to claim 47or claim 48 in combination with a pharmaceutical excipient.
 51. Thepharmaceutical preparation of claim 50, wherein the excipient is asugar.
 52. The pharmaceutical preparation of claim 50, in lyophilizedform.
 53. The pharmaceutical preparation of claim 50, further comprisinga liquid diluent.
 54. The pharmaceutical preparation of claim 53,wherein the liquid diluent is selected from the group consisting ofbacteriostatic water for injection, dextrose 5% in water,phosphate-buffered saline, Ringer's solution, saline solution, sterilewater, deionized water, and combinations thereof.
 55. The pharmaceuticalpreparation of claim 50, in unit dosage form.
 56. The pharmaceuticalpreparation of claim 50, housed in a glass vial.
 57. A method ofdelivering a conjugate comprising the step of administering to a patienta therapeutically effective amount of a conjugate according to claim 47or claim
 48. 58. A gel comprising a water-soluble polymer of any one ofclaims 1-46.
 59. A method of preparing a water-soluble polymer having animide branching moiety comprising the steps of: (i) providing a firstwater-soluble polymer segment carrying a first reactive group, the firstreactive group comprising a carbonyl group; (ii) reacting the firstwater-soluble polymer segment with ammonia to form a water-solublepolymer intermediate carrying an amide group (—C(O)—NH₂); (iii) treatingthe water-soluble polymer intermediate with a strong base to remove aproton from the terminal amine of the amide group; (iv) reacting thewater-soluble polymer intermediate with a second water-soluble polymersegment carrying a second reactive group comprising a carbonyl group toform an imide-linked water-soluble polymer, the imide linking groupcomprising a linking nitrogen atom, a first carbonyl group covalentlyattached to the linking nitrogen atom, and a second carbonyl groupcovalently attached to the linking nitrogen atom; and (v) optionally,attaching a reactive group to the linking nitrogen atom of theimide-linked water-soluble polymer.
 60. The method of claim 59, whereinsaid attaching step (v) comprises treating the imide-linkedwater-soluble polymer with a strong base to remove a proton from thelinking nitrogen atom and reacting the imide-linked water-solublepolymer with a molecule having the structure Z-(X₃)_(c)-Q, wherein Z isa reactive group, (c) is either zero or one, X₃, when present, is aspacer moiety, and Q is a leaving group.
 61. The method of claim 60,wherein Z is selected from the group consisting of hydroxyl (—OH),ester, orthoester, carbonate, acetal, aldehyde, aldehyde hydrate,ketone, vinyl ketone, ketone hydrate, thione, thione hydrate, hemiketal,sulfur-substituted hemiketal, ketal, alkenyl, acrylate, methacrylate,acrylamide, sulfone, amine, hydrazide, thiol, thiol hydrate, carboxylicacid, isocyanate, isothiocyanate, maleimide

succinimide

benzotriazole

vinylsulfone, chloroethylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates,thiosulfonate, tresylate, and silane.
 62. The method of claim 60,wherein Q is halo or a sulfonate ester.
 63. The method of claim 60,wherein Q is mesylate ester or tosylate ester.
 64. The method of claim60, wherein X₃ is selected from the group consisting of a bivalentcycloalkyl group comprising 3 to about 20 carbon atoms, an amino acid, aC1-C10 alkylene group, —(CH₂)_(o)—Y—(CH₂)_(p)— wherein (o) and (p) areeach independently zero to about 10 and Y is selected from the groupconsisting of —O—, —S—, —N(R₆)— wherein R₆ is H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl and substitutedaryl, —C(O)—, —C(S)—, —C(O)—O—, —C(O)—NH—, —NH—C(O)—NH—, and—O—C(O)—NH—, and combinations thereof, wherein X₃ may optionally furtherinclude an ethylene oxide oligomer chain comprising 1 to 20 ethyleneoxide monomer units.
 65. The method of claim 59, wherein each of thefirst and second water-soluble polymer segments is a poly(ethyleneglycol).
 66. The method of claim 65, wherein each poly(ethylene glycol)comprises an alkoxy end-capping moiety.
 67. The method of claim 59,wherein the first and second reactive groups are carbonate containingreactive groups having the structure —O—C(O)—O—X_(r), wherein X_(r) isaryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle,or substituted heterocycle.
 68. The method of claim 67, wherein X_(r) is1-benzotriazolyl, o-, m-, or p-nitrophenyl, or N-succinimidyl.
 69. Themethod of claim 59, wherein the imide-linked water-soluble polymer hasthe structure:

wherein: POLY₁ is a first water-soluble polymer segment; POLY₂ is asecond water-soluble polymer segment; each of (a) and (b) isindependently either zero or one; X₁, when present, is a first spacermoiety; and X₂, when present, is a second spacer moiety.
 70. The methodof claim 59, wherein, following said attaching step (v), theimide-linked water-soluble polymer has the structure:

wherein: POLY₁ is a first water-soluble polymer segment; POLY₂ is asecond water-soluble polymer segment; each of (a), (b) and (c) isindependently either zero or one; X₁, when present, is a first spacermoiety; X₂, when present, is a second spacer moiety; X₃, when present,is a third spacer moiety; and Z is a reactive group.
 71. The method ofclaim 59, further comprising the step of recovering the imide-linkedwater-soluble polymer.
 72. The method of claim 71, wherein therecovering step is carried out by chromatography.
 73. The method ofclaim 59, further comprising the step of purifying the imide-linkedwater-soluble polymer.
 74. A method of preparing a water-soluble polymerhaving an imide branching moiety comprising the steps of: (i) providinga first water-soluble polymer segment carrying a first reactive group,the first reactive group comprising a carbonyl group; (ii) reacting thefirst water-soluble polymer segment with a molecule comprising a secondreactive group and an amine group, optionally separated by a spacermoiety, to form a water-soluble polymer intermediate comprising an amidelinkage (—C(O)—NH—) between the water-soluble polymer segment and thesecond reactive group; (iii) treating the water-soluble polymerintermediate with a strong base to remove a proton from the nitrogenatom of the amide linkage; and (iv) reacting the water-soluble polymerintermediate with a second water-soluble polymer segment carrying athird reactive group comprising a carbonyl group to form an imide-linkedwater-soluble polymer, the imide linking group comprising a linkingnitrogen atom, a first carbonyl group covalently attached to the linkingnitrogen atom, and a second carbonyl group covalently attached to thelinking nitrogen atom.
 75. The method of claim 74, wherein the moleculecomprising a second reactive group and an amine group has the structureZ-(X₃)_(c)—NH₂, wherein Z is the second reactive group, (c) is eitherzero or one, and X₃, when present, is a spacer moiety.
 76. The method ofclaim 75, wherein Z is selected from the group consisting of hydroxyl(—OH), ester, orthoester, carbonate, acetal, aldehyde, aldehyde hydrate,ketone, vinyl ketone, ketone hydrate, thione, thione hydrate, hemiketal,sulfur-substituted hemiketal, ketal, alkenyl, acrylate, methacrylate,acrylamide, sulfone, amine, hydrazide, thiol, thiol hydrate, carboxylicacid, isocyanate, isothiocyanate, maleimide

succinimide

benzotriazole

vinylsulfone, chloroethylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates,thiosulfonate, tresylate, and silane.
 77. The method of claim 75,wherein X₃ is selected from the group consisting of a bivalentcycloalkyl group comprising 3 to about 20 carbon atoms, an amino acid, aC1-C10 alkylene group, —(CH₂)_(o)—Y—(CH₂)_(p)— wherein (o) and (p) areeach independently zero to about 10 and Y is selected from the groupconsisting of —O—, —S—, —N(R₆)— wherein R₆ is H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl and substitutedaryl, —C(O)—, —C(S)—, —C(O)—O—, —C(O)—NH—, —NH—C(O)—NH—, and—O—C(O)—NH—, and combinations thereof, wherein X₃ may optionally furtherinclude an ethylene oxide oligomer chain comprising 1 to 20 ethyleneoxide monomer units.
 78. The method of claim 74, wherein each of thefirst and second water-soluble polymer segments is a poly(ethyleneglycol).
 79. The method of claim 78, wherein each poly(ethylene glycol)comprises an alkoxy end-capping moiety.
 80. The method of claim 74,wherein the first and third reactive groups are carbonate containingreactive groups having the structure —O—C(O)—O—X_(r), wherein X_(r) isaryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle,or substituted heterocycle.
 81. The method of claim 80, wherein X_(r) is1-benzotriazolyl, o-, m-, or p-nitrophenyl, or N-succinimidyl.
 82. Themethod of claim 74, wherein the imide-linked water-soluble polymer hasthe structure:

wherein: POLY₁ is a first water-soluble polymer segment; POLY₂ is asecond water-soluble polymer segment; each of (a), (b) and (c) isindependently either zero or one; X₁, when present, is a first spacermoiety; X₂, when present, is a second spacer moiety; X₃, when present,is a third spacer moiety; and Z is a reactive group.
 83. The method ofclaim 74, further comprising the step of recovering the imide-linkedwater-soluble polymer.
 84. The method of claim 83, wherein therecovering step is carried out by chromatography.
 85. The method ofclaim 74, further comprising the step of purifying the imide-linkedwater-soluble polymer.